Currently, one of the most interesting concerns in the life and medical sciences is the systemic analysis of proteins involved in diseases because they can be used in the treatment and prevention of the diseases.
With the great advances in fundamental sciences that contribute to drug discovery, including molecular biology and genomics (genome science), the category of drug discovery has expanded and changed rapidly. Particularly, genomic drugs, typifying new drugs, require new and different methods for the discovery thereof.
To be developed into or used as new drugs, newly discovered substances must be proven to have physiological activity with respect to specific diseases or under specific conditions, which is a task for the life and medical sciences. Most of these biologically active substances are composed of proteins, and thus the determination of protein structure and function falls under the category of the life and medical sciences.
It was estimated that there are about 35,000 human genes. However, the number of proteins produced from the genome is estimated to amount to hundreds of thousands to millions. Because proteins are responsible for almost all reactions occurring in cellular organelles, the systemic study of gene functions at the protein level is needed.
A protein is a complex described with reference to various properties including molecular weight, isoelectric point (pI), hydrophilicity, hydrophobicity, etc. For the analysis of a protein of interest, therefore, it must be isolated as a pure form, followed by identifying it through the use of, for example, mass analysis or bioinformatics. As for the proteins involved in diseases, they are generally analyzed using high performance isolation techniques because their amounts are smaller than those of general proteins.
Proteins, which play an essential role in life phenomena, function alone or via interaction with other molecules including proteins, DNA, synthetic compounds, photons, etc. The mere description of the physical and/or chemical properties of a protein does not indicate a complete understanding thereof. In order to comprehend a protein of interest, not only must the molecules interacting therewith be identified, the interaction modality (physiological activity) also has to be revealed. That is, for a complete understanding of a protein of interest, how the protein of interest interacts, and with which molecules, needs to be determined.
Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a typical method of analyzing proteins. In this method, proteins are separated on a polyacrylamide gel plate to which an electric field is applied. The SDS-PAGE is an one-dimensional method widely used in the simple separation and identification of a protein of interest on the basis of molecular weight. This technique suffers from disadvantages in that proteins in SDS solutions are denatured, and thus have tertiary structures different from the native structures thereof, and proteins are trapped within the gel.
With the one-dimensional technique alone, it is difficult to separate and discern the mixture of numerous different proteins comprising a proteome, which is defined as the entire complement of proteins expressed by a genome, the production and relative amounts of individual proteins thereof varying depending on the physiological conditions of cells or tissues.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is commonly used to separate complex mixtures of proteins based on two properties thereof, typically iso-electric point and molecular weight [Thou, F.; Johnston, M. V. Anal. Chem. 2004, 76, 2734-2740; Klose, J.; Kobalz, U. Electrophoresis, 1995, 16, 1034-1059; Righetti, P. G.l Castagna, A.; Herbert, B. Prefractionation Techniques in Proteome Analysis, Anal. Chem. 2001, 73, 320A-326A.].
In 2D-PAGE, the proteins are first separated on the basis of their isoelectric points (pI). In the presence of an electric field, proteins migrate on flat bed immobilized pH gradient gel strips based on an ampholyte and focused at their isoelectric points (isoelectric focusing IEF). It typically takes about 12 hours or longer to complete IEF.
Next, the strips, on which the proteins are immobilized at pI, are placed on a vertical SDS-PAGE slab gel and electrophoresed to separate proteins on the basis of their molecular weight. Proteins with smaller molecular weights migrate farther toward the bottom of the vertical slab. The total time period during which 2D-PAGE is completely conducted amounts to about 36 hours.
Following the completion of 2D-PAGE, staining the gel reveals the positions of individual proteins as spots or smudges. Optionally, these proteins may be recovered and subjected to enzyme digestion for mass spectrometry analysis.
2D-PAGE is a high resolution tool and as such can effectively determine the general pattern of proteins. Allowing the separation of proteins on a semi-preparative level, 2D-PAGE can be applied for the analysis of very complex human plasma proteins as well as proteins extracted from urine and tissues, which leads to the detection and diagnosis of diseases [(Giddings, J. C., Unified Separation Science, John Wiley & Sons, New York 1991, pp. 126-128.].
However, 2D-PAGE is a labor-intensive technique, and is not only difficult to automate, but also has limitations in detection sensitivity and dynamically active range. Also, proteins are separated as denatured forms due to the SDS solution used in 2D-PAGE. Furthermore, the separated proteins trapped within the gel matrix are difficult to recover. Thus, the proteins are enzymatically digested within the gel and recovered in peptide forms before analysis.
Capillary isoelectric focusing is a high-resolution technique for protein separation based on differences in isoelectric points (pI) using the silica capillary, in which a pH gradient is formed by filling a solution of ampholytes, followed by applying an electric field thereto [Conti, M.; Gelfi, C.; Righetti, P. G. Electrophoresis 1996, 17, 1485-1491.].
CIEF is identical in fundamental focusing principle to gel-based IEF, but there is a difference therebetween in terms of the place where focusing is conducted. Because it is accomplished within a silica capillary, CIEF can be used to analyze a small amount of proteins. Thanks to its high sensitivity, the capillary allows proteins to be separated even if their pI values differ by as little as 0.003 [Quigley, W. C.; Dovichi, N. J. Anal. Chem. 2000, 76, 4645-4658].
However, the capacity of CIEF is insufficient to treat a complex mixture of proteins, such as a proteome, and thus an attempt has recently been made to conduct CIEF in association with a secondary separation technique, such as chromatography, rather than alone, in order to enhance synergic separation efficiency.
Reverse phase liquid chromatography (RPLC) is a representative secondary separation technique that can accomplish two-dimensional separation in conjunction with CIEF on-line. In CIEF-RPLC, proteins are separated by pI, followed by secondary separation on the basis of hydrophobicity in a chromatography column [Chen, J.; Lee, C. S.; Shen, Y.; Smith, R. D.; Baehrecke, E. H. Electrophoresis 2002, 23, 3143-3148.]. When applied to the peptide mixture hydrolyzed from drosophila proteome, this technique afforded 1,800 peaks of proteins for 8 hours.
CIEF-CGE, in which capillary isoelectric focusing is on-line coupled with capillary gel electrophoresis, uses a capillary tube filled with polyacrylamide gel instead of a polyacrylamide gel plate to separate proteins on the basis of molecular weight, and has been applied for the separation of simple proteins such as hemoglobin [Yang, C.l Liu, H.; Yang, Q.; Zhang, L.; Zhang, W.; Zhang Y. Anal. Chem. 2003, 75, 215-218.].
CIEF-RPLC is useful in separating a mixture of peptides rather than proteins. This technique is difficult to apply to proteins because their chains are broken and lost upon passage through the column. CIEF-RPLC may be further coupled with electrospray ionization (ESI) mass spectrometry [Tnag, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1996, 68, 2482-2487; Yang, L.; Lee, C. S.; Hofstadler, S. A.; Papsa-Toli, L.; Smith, R. D. Anal. Chem. 1998, 70, 3235-3241; Martinovi, S.; Berger, S. J.; Papsa-Toli, L.; Smith, R. D. Anal. Chem. 2000, 72, 5356-5360]. However, this combination suffers from the disadvantage of requiring an additional purification process for removing the ampholytes used in the CIEF after the completion of separation.
Therefore, unless the ampholytes are removed in advance, it is difficult to perform a sample analysis due to the interruption of ions in the solution. In order to avoid this problem, a microdialysis membrane, such as anion cells, is used to remove a significant amount of the ampholytes [Zhou, F.; Johnston, M. V. Anal. Chem. 2004, 76, 2734-2740]. This technique, although able to separate proteins, cannot avoid the denaturation of proteins due to the organic solvent used in RPLC, and is still inapplicable to the separation of large molecular weight proteins.
Among the techniques for protein separation on the basis of molecular weight is flow field flow fractionation (FlFFF), a subtechnique of flow field fraction (FFF). This technique is a new analytical tool, developed primarily for the separation of macromolecules and particles, such as proteins, cells, water-soluble polymers and nanoparticles and the characterization thereof for size, diffusion coefficient, molecular weight, etc. [J. C. Giddings, F. J. F. Yang, M. N. Myers, Science 1976, 193, 1976, 1244; M. H. Moon, P. S. Williams, H. Kwon, Anal. Chem. 1999, 71(14), 1999, 2657.].
Generally, FFF employs a hollow rectangular parallelepiped channel filled with a flowing phase. As a liquid flow initiates along the channel axis, samples migrate and separate depending on the strength of the external field applied thereto in a direction perpendicular to the liquid flow. In flow field-flow fractionation (FlFFF), the external field is a cross-flow of a carrier liquid, perpendicular to the usual channel flow. The field strength, which is a critical factor for controlling the retention of macromolecules, such as proteins, is thus determined by the flow rate of this cross-flow.
Across the channel of FlFFF, there is no net flux; that is, the retention of the sample is at equilibrium between the flow rate of the cross-flow at the bottom and the Brownian diffusion motion. The mean height along which samples migrate is determined by the extent of Brownian diffusion, which depends on the molecular weights, or Stoke's size in the case of proteins. Hence, samples with lower molecular weights remain at higher positions from the channel bottom with an increasing flow rate of the cross-flow as they diffuse greater distances. The flow profile between the two parallel walls is parabolic, with the highest flow velocity located near the center of the channel and flow velocity decreasing towards the walls. Under these circumstances, proteins or macromolecular samples can be separated on the basis of size. That is, samples with smaller molecular weights are allowed to flow out of the channel with greater precedence.
In another technique for protein separation and analysis, a hollow fiber membrane is utilized as a separation channel [W. J. Lee, B.-R. Min, M. H. Moon, Anal. Chem. 1999, 71(16), 3446; M. H. Moon, K. H. Lee, B.-R. Min, J. Microcolumn September, 1999, 11(9), 676; P. Reschiglian, A. Zattoni, D. Parisi, L. Cinque, B. Roda, F. D. Piaz, A. Roda, M. H. Moon, B.-R. Min, Anal. Chem. 2005, 77, 47]. In this technique, the role of an external field is determined by the flow rate of the cross-flow or radial flow discharged into the outer wall of the hollow fiber membrane. Within the channel, the samples migrate, forming a circular band at equilibrium against the external field, as shown in FIG. 1. The separation of samples can be accomplished by controlling the flow rate of the liquid flowing along the axis of the channel.
FIG. 1 is a schematic diagram showing the structure of an apparatus for protein separation using hollow fiber flow field flow fractionation (HFFlFFF). Liquid is transferred into the hollow fiber by an HPLC pump 140, and protein samples eluted from the channel are detected using a UV/VIS detector 130.
Hollow fiber flow field flow fractionation can separate proteins in increasing order of molecular weight as well as in non-denatured forms, thanks to the use of a buffer as a carrier. Further, this technique enjoys the advantages of avoiding the destruction of proteins and the plugging of the channel because the channel is not filled with ampholytes. Moreover, in the case where the inner diameter of the hollow fiber membrane is narrowed, even a trace amount of proteins may be used for analysis with this technique [I. Park, K.-J. Paeng, D. Kang, M. H. Moon, J. Separation Sci., 2005, 28, 2043; D. Kang, M. H. Moon, Anal. Chem., 2005, 77, 4207].
In addition to separating proteins in order from low molecular weight to high molecular weight and avoiding the destruction of protein samples or the plugging of the hollow fiber membrane channel, hollow fiber flow field-flow fractionation is also advantageous in terms of economy, because the hollow fiber membrane is relatively inexpensive. However, this method is not as high in resolution power as is required, and is incapable of separation on the basis of various protein properties.